Cardiac muscle contraction is initiated by morphological changes in the contractile proteins of the cardiac myofibrils. Phosphorylation of myosin light chain 2 (MLC2) and other contractile proteins (troponin T (TnT) and troponin I (TnI)) by protein kinase C (PKC) isozymes and myosin light chain kinase (MLCK) is a vital step that leads to these changes. Previous studies on the regulation of muscle contraction focused mainly on skeletal and smooth muscles. The relevance ofMLC2 phosphorylation by MLCK and PKC isozymes in cardiac function is still unclear. Our long range goal is to identify the functions of site-specific phosphorylation of myosin light chain 2 in cardiac muscle biology. The objective of this proposal is to understand the biochemical and physiological characteristics of site-specific phosphorylation of MLC2 by PKC and MLCK. Our central hypothesis is that PKC and MLCK phosphorylate MLC2 at specific sites and that the phosphopeptide in each site has a specific function in cardiac physiology. Additionally, we hypothesize that MLC2 phosphorylation affects myofibrillar MgATPase activity and calcium sensitivity of the myofibrils differently. These hypotheses are formulated based on the strong preliminary data generated in our laboratory (see figs 1-10). These hypotheses will be tested and our objectives achieved by (1) determining the sites of in vitro and in situ phosphorylation of MLC 2 by PKC and MLCK, and comparing the amino acid sequences of the phosphopeptides generated, (2) determining the functional relevanvce of MLC2 phosphopeptide at each major phosphorylation site and (3) measuring the contractile movement of in situ phosphorylated cardiac myocytes isolated from bovine ventricles. Our rationale for this proposal is that the knowledge of the amino acid sequence of the phosphopeptides and the functional significance of site specific phosphorylation will provide new strategies that can be used to prevent, regulate or treat cardiovascular diseases. This proposal is very innovative because it uses molecular, biochemical and physiological approaches to reveal the functional importance of site-specific phosphorylation. We have the expertise, experience and the required research environment to successfully accomplish the proposed work based on our past and ongoing contributions in cardiovascular research. The following outcome is anticipated. (a) Enhancement of the understanding of the molecular and physiological relevance of MLC2 phosphorylation in cardiovascular biology (b) Phosphorylation sites generated will reveal the preferential sites phosphorylated by PKC and MLCK and their relative site-specific functions. These outcome are significant because it is expected that the knowledge gained from this study will make fundamental new contributions to the field of cardiovascular physiology and cardiomyopathy.